1. Field of the Invention
This invention relates to members of the transforming growth factory-xcex2 family and their regulation of cell division, cell survival, and the specification of cell fates. Particularly, the invention relates to the bone morphogenetic protein (BMP)-2/4 homolog decapentaplegic (dpp) and its role in the maintenance of stem cells. For example, a dpp-based method for maintenance and controlling the division of germline stem cells, and a dpp-based method for defining a niche that controls germline stem cell proliferation are disclosed. Additionally, the invention provides a model of ovarian tumor development. The invention further relates to a dpp-based method for propagating stem cells in an undifferentiated state in vivo or by culturing in vitro.
2. Description of Related Art
In many adult tissues that undergo continuous cell turnover, a population of stem cells is responsible for replacing lost cells. Because of their pivotal role in controlling growth and neoplasia, the mechanisms regulating stem cell function are of great interest (reviewed by Potter and Loeffler, 1990; Doe and Spana, 1995; Lin, 1997; Morrison et al., 1997). Two mechanisms have been proposed to maintain stem cell divisions and regulate the differentiation of stem cell daughters: intrinsic factors and extracellular signals. Asymmetrically localized intrinsic factors help specify the fates of neuroblast daughters in Drosophila embryos (Doe and: Spana, 1995). Extracellular signals from surrounding cells mediated by cell surface-associated ligands and diffusible factors are frequently involved (Potter and Loeffler, 1990; Morrison et al., 1997). The identification of several of these factors has made it possible to culture some types of stem cell in vitro.
The Drosophila ovary presents an excellent system for studying two distinct groups of stem cells that remain active during much of adult life (reviewed by Spradling et al., 1997). The adult ovary contains 14-16 ovarioles each with a germarium at the tip, within which the germline and somatic stem cells are located. Two or three germline stem cells, located at the anterior tip of the germarium, divide asymmetrically to generate all germline cells in the ovariole (Wieschaus and Szabad, 1979; reviewed by Lin, 1997). Stem cell daughters known as cystoblasts undergo four rounds of synchronous division to produce groups of two, four, eight, and eventually 16 interconnected cystocytes, the precursors of ovarian follicles (reviewed by de Cuevas et al., 1997). Two somatic stem cells residing in the middle of the germarium give rise to all the somatic follicle cells (Margolis and Spradling, 1995); their equivalent in the testis are cyst progenitor cells. Three types of mitotically quiescent somatic cells are located in the vicinity of the stem cells: terminal filament and cap cells contact the germline stem cells, while inner sheath cells lie more posteriorly and contact both stem cell types.
Germline stem cell division is known to involve intrinsic mechanisms. This division and subsequent cystocyte divisions are physically unequal due to the segregation of fusomes rich in membrane skeleton proteins such as xcex1-spectrin and an adducin homolog encoded by hu-li tai shao (hts) (reviewed by de Cuevas et al., 1997). The round fusome (or xe2x80x9cspectrosomexe2x80x9d) characteristic of stem cells changes shape as cyst development proceeds, allowing cysts at different stages to be identified. The bag of marbles (bam) gene is highly expressed only in the stem cell daughter (McKearin and Spradling, 1990). The loss of Bam protein in cystoblasts prevents their differentiation, causing germline tumors to form (a xe2x80x9ctumorxe2x80x9d in Drosophila is a large clump of proliferating cells, the term does not imply these cells are cancerous). The genes pumilio (pum) and nanos (nos), encoding translational regulators, also play critical roles in the formation and maintenance of germline stem cells (Lin and Spradling, 1997; Forbes and Lehmann, 1998).
Less is known about the intercellular signals that control stem cell proliferation. Two important signaling molecules, Hedgehog (Hh) and Wingless (Wg) (reviewed by Perrimon, 1995; Cadigan and Nusse, 1997), are expressed in terminal filament and cap cells (Forbes et al., 1996a and 1996b). Hh signaling is critical for proliferation and differentiation of follicle cells, but it remained to be determined at the time the present invention was made whether somatic stem cells or their daughters are regulated (Forbes et al., 1996a and 1996b). The role of these signals in the germ line was even less clear because ectopic expression of hh did not appear to interfere with the function of germline stem cells (Forbes et al., 1996a).
Members of the transforming growth factor-xcex2(TGF-xcex2) family, including TGF-xcex2s, activins, and the bone morphogenetic proteins (BMPs), elicit a broad range of cellular responses including the regulation of cell division, survival, and specification of cell fates (reviewed by Massague et al., 1996; Hogan, 1996a). TGF-xcex2s were previously identified as repressing the proliferation of stem cells as assayed by either in vitro cultures or in vivo ectopic expression (Potter and Leoffler, 1990; Morrison et al., 1997). Inactivation of BMP-4 and its receptor BMPR in mice resulted in embryonic lethality for homozygous mutants (Winnier et al., 1995; Mishina et al., 1995), but no effect on stem cells was noted.
Similarly dpp, encoding a vertebrate BMP-2/4 homolog in Drosophila, functions as a local signal as well as a long-distance morphogen to pattern the early embryo and adult appendages by regulating cell proliferation and cell fate determination (Padgett et al., 1987; reviewed by Lawrence and Struhl, 1996). dpp is expressed in an anterior subset of follicle cells, and is required for establishing egg shape and polarity during late stages of oogenesis (Twombly et al., 1996). But an effect of dpp on maintaining and propagating stem cells, instead of causing their differentiation, has not been previously shown.
Major participants in the dpp signaling pathway have been identified: saxophone (sax) and thick veins (tkv) encode type I serine/threonine kinase transmembrane receptors, whereas punt encodes a type II serine/threonine kinase transmembrane receptor (Brummel et al., 1994; Nellen et al., 1994; Penton et al., 1994; Xie et al., 1994; Ruberte et al., 1995; Letsou et al., 1995). mothers against dpp (mad), Medea (Med), and Daughters against dpp (Dad) encode a family of conserved TGF-xcex2 transducers (Sekelsky et al., 1995; Tsuneizumi et al., 1997; Hudson et al., 1998; Wisotzkey et al., 1998; Das et al., 1998; Inoue et al., 1998), collectively known as Smads. Smads are proteins which transduce signals on behalf of TGF-xcex2 family members, or inhibit TGF-xcex2 signal transduction. A paradigm for TGF-xcex2 signal transduction has been developed from several experimental systems (Heldin et al., 1997). In Drosophila, Dpp binds both type I and II receptors to allow the constitutively active Punt kinase to phosphorylate and activate type I kinases, which phosphorylate Mad. The phosphorylated Mad brings Med into the nucleus as a transcriptional activator to stimulate dpp target gene expression.
Enhancing Dpp or other BMP-like signaling activities can be achieved by reducing the presence of Dad-like proteins, such as human Smad6 and Smad7. Vertebrate Smad6 and Smad 7 interact with type I receptors, and are known to inhibit both TGF-xcex2 and BMP signaling in cultured cells and frog embryos. Thus, disinhibition of TGF-xcex2 family members by inhibiting certain Smads promotes BMP-like signaling cascades. Additionally, Dpp or other BMP-like signaling activities may be increased by enhancing the function of Dpp or BMP receptors, such as Sax, Tkv, and Punt in Drosophila, and BMP receptors BMPR-II, ActR-II, Act-IIB, BMPR-IA, and ActR-I in humans. Other downstream positive regulators of Dpp or BMP signaling include Mad, Med, Dad, and Schnurri proteins in Drosophila, and Smad1, Smad4 and Smad5 in humans. See review by Padgett (1999).
Therefore, to address the prior art""s failure to identify and characterize factors involved in germline stem cell maintenance and propagation, we now disclose that a member of the TGF-xcex2 family of growth factors and its signaling pathway unexpectedly provide this essential function.
It is an object of the invention to maintain and/or propagate stem cells by stimulating signaling through a bone morphogenetic protein (BMP) signaling pathway. In this manner a population of stem cells can be maintained in vivo or in vitro, and/or expanded.
Methods for maintaining germline and somatic stem cells of an organism are provided by stimulating a bone morphogenetic protein (BMP) signaling pathway.
The signal transduction pathway associated with a BMP specifically binding to a receptor may involve phosphorylation of serine/threonine residues (e.g., kinases, phosphatases) and a cascade of components of the pathway (i.e., signal transducers such as, for example, transcription factors) which communicate that signal. For example, a signal may be communicated from BMP binding at the cell surface to the nucleus where gene expression of downstream targets are either activated or inhibited. Thus, BMP signaling may be modulated at one or more steps in this pathway, or by affecting upstream regulators or downstream targets of this signaling pathway. Modulation (i.e., stimulation or repression) of BMP signaling may be accomplished directly on the stem cell or indirectly through other cells in a mixed cell population (e.g., feeder layer).
Properties of the stem cell may be maintained by stimulating BMP signaling. Furthermore, stem cells may be increased in abundance and/or increased in lifetime by such stimulation. Conversely, stem cells or tumor cells in a population may be reduced in total number or concentration, or even eliminated at the limit of detection, by repressing BMP signaling.
Stem cells may also be propagated and isolated according to the invention.
Our invention addresses the problem of restricted access to and limited numbers of stem cells. The ability to maintain and to propagate stem cells facilitates genetic manipulation and the characterization of these rare cells.
The present invention provides a method for maintaining and controlling the division of germline stem cells in which dpp can provide an essential role. Further, it provides a model of ovarian tumor formation in which overexpression of dpp produces ovarian stem cell tumors. Clonal analysis demonstrates that downstream components (i.e., signal transducers) of the dpp signaling pathway are required cell-autonomously in the germline stem cells for their division and maintenance. This invention also provides a method for control of a cellular niche by BMP signaling, in which germline stem cells are regulated by, for example, a dpp signal that likely derives from surrounding somatic cells.
Stem cells are thought to be regulated by positive and negative diffusible factors, but the functions of most of these factors have never been demonstrated in vivo. The present invention provides a method in which Dpp directly signals to maintain Drosophila germline stem cells and stimulate their division. The experiments of the examples were made possible by a clonal cell marking method that allows the function of stem cells and their progeny to be examined directly over many cell generations. In addition to the dpp signal, known components in the dpp signal transduction pathway were shown to be required in these adult stem cells. This action appears to be specific to stem cells, since germ cells lacking dpp pathway components were still able to form 16-cell cysts. The examples demonstrate that a TGF-xcex2-like molecule functions as a stem cell growth factor.
dpp signal transduction is required for maintaining stem cells, on which Dpp may act in several distinct ways. Signaling prevents germline stem cells from differentiating into cystoblasts and gametes. The examples show that overexpressed dpp prevents stem cell differentiation, while reduction of dpp function promotes stem cell differentiation. An attractive candidate target of the dpp signal transduction pathway is the Bam protein, which is normally synthesized at much higher levels in cystoblasts than in stem cells (McKearin and Ohlstein, 1995). The forced expression of Bam in germline stem cells causes them to differentiate in a manner very similar to that caused by reductions in dpp signaling (Ohlstein and McKearin, 1997). Thus, dpp signaling may negatively regulate Bam protein levels in germline stem cells. Two other genes, pum and nos, are required to form and maintain germline stem cells (Lin and Spradling, 1997; Forbes and Lehmann, 1998). In the embryo, both proteins work together to repress the translation of target genes such as hunchback (hb) (Baker et al., 1992; Murata and Wharton, 1995). In the ovary, dpp signaling may downregulate Bam through effects on the Nos/Pum pathway or by an independent mechanism. However, genes throughout the dpp pathway are required, including two nuclear transcription factors, suggesting that the action of the pathway is on transcription of target genes. Also see reviews by Attisano and Wrana (1998), Kawabata et al. (1998), and Padgett et al. (1998).
dpp may also function to maintain a specialized association between the stem cells and basal terminal filament cells. Such an association has been postulated to hold the stem cells at the anterior of the germarium, while daughter germline cells all move posteriorly and eventually leave the germarium. The results presented herein indicate that the stem cell loss is due to differentiation. Possibly, dpp signaling via its receptor regulates the expression of adhesion molecules that reside on the cell surface or of cytoplasmic proteins that indirectly promote stem cell adhesion.
dpp signaling also may act to stimulate stem cell division. dpp signaling stimulates cell proliferation at several points during Drosophila development. In the wing imaginal disc, it is essential for cell proliferation and/or survival (Burke and Baster, 1996), whereas it promotes the G2-M transition in the morphogenetic furrow of the developing eye disc (Penton et al., 1997). Consistent with such a requirement, mad mutants have greatly reduced imaginal discs, shortened gastric caeca, and small brains (Sekelsky et al., 1995). The requirement for dpp signaling disclosed herein suggests that adult stem cells use strategies similar to those of embryonic and larval somatic cells to regulate proliferation. For example, dpp stimulates the rate of cell division for stem cells.
During aging, the number and activity of stem cells are thought to be reduced. The examples indicate that the level of dpp signaling controls the life span and division rate of germline stem cells. Reduced dpp signaling caused premature stem cell loss. Perhaps more surprising is the observation that putative increases in signaling, caused by removal of Dad protein activity from stem cells, permitted stem cells to be maintained longer. This finding suggests that dpp signaling not only is necessary, but may sometimes be rate limiting for stem cell maintenance. The illustrative examples demonstrate for the first time a method in which stem cell life span has been extended in an intact organism.
These results suggest that it may be possible to extend the life span of stem cells, a process that could be of therapeutic significance. For example, drugs that upregulate BMP signaling to stem cells may enhance fertility in humans and animals, such as male fertility in patients with reduced numbers of germline stem cells (basal cells). Such drugs may ameliorate hematologic conditions caused by reduced stem cell functioning, for example aplastic anemias, agammaglobulinemia, and related conditions. Drugs enhancing BMP signaling may enhance wound healing. Aging-related pathologies caused by loss of stem cells, such as hair loss, loss of muscle mass, reduction of blood cell numbers, and the aging of the skin and other stem cell-dependent tissues could be treated by increasing BMP signal transduction. Compounds enhancing BMP signaling may increase the average lifespan of an organism.
One method for the enhancement of dpp signal transduction may be facilitated by removal of the dpp inhibitor Dad or other Dad-like inhibitory protein activity (inhibitory Smad activity) from the germline stem cells. Dad is induced by dpp signaling, but then acts to dowregulate the very pathway that activated its production. This method could also be practiced with other negative regulators of the dpp signaling pathway and, in particular, inhibitory Smads. In contrast, brinker (brk) is a target gene repressed by dpp signaling and, because it is itself a transcriptional repressor, the net effect of repressing expression of the Brk repressor is to upregulate Brk-regulated target genes (Minami et al., 1999; Campbell et al., 1999; Jazwinska et al., 1999). This results in the increased production of Brk-regulated target genes following dpp signaling. Hence, BMP signaling can be stimulated or repressed by appropriate manipulation of Smads or target genes which are regulated by BMP signaling (i.e., increasing or decreasing their effects as appropriate to achieve stimulation or repression of BMP signaling). The roles of Dad and Brk, like the rest of the pathway, appear to be conserved in mammals.
Drugs that inhibit BMP signaling to stem cells may be useful chemotherapeutic agents. For example, drugs inhibiting BMP signaling pathways may be useful therapies against teratocarcinoma by causing stem cell differentiation. As another example, drugs which inhibit BMP signaling may be successful treatments against ovarian germline tumors dependent upon BMP signaling for continued growth.
Increased or decreased BMP signaling to stem cells might allow populations of stem cells to expand prior to bone marrow transplant, thereby increasing the chances of successful transplantation and reducing the amount of donor marrow required. Further, control of BMP signaling pathways may permit stem cells other than those in bone marrow to be removed from a patient, expanded in vitro, and subsequently reintroduced in to the patient to repair tissues damaged by injury or disease, such as Parkinson""s disease.
Bone marrow from patients with hematologic tumors, such as lymphoma and leukemia, could be tested for BMP sensitivity. Positive test results for BMP sensitivity would allow steps to be taken to avoid potential side effects of anti-BMP treatment in vivo. For example, marrow removed from the patient could be cleansed of tumors cells by inhibiting BMP signaling, thereby inducing differentiation of tumor cells and reducing the tumor burden. The cleansed marrow would subsequently be returned to the patient in an autologous bone marrow transplant. Such differentiation therapy could also be used for solid tumors like sarcoma, carcinoma, and neuroglioma to reduce tumor burden. Therapy may be use alone or in association with other treatments such as, for example, chemotherapy, hyperthermia, or radiation which preferentially kills rapidly dividing cells and surgical resection of tumor.
Upregulation of BMP signaling to stem cells may permit the growth of germline stem cells in culture, useful in, for example, generating transgenic animals. Such techniques are especially useful in organisms which have not traditionally been used as genetic models of development and disease.
The ability to expand stem cell niches by overexpression of TGF-xcex2 members, such as dpp may allow rare human stem cells, or alternatively rare stem cells of any species, to be purified and propagated following transfer into living Drosophila, which have been genetically engineered to serve as hosts.
Beside biomedical research and treatment, other uses for the present invention include agriculture and wildlife conservation. Stem cells could be provided in or obtained from humans, primates (e.g., bonobo, chimpanzee, gorilla, macaque, orangutan), companion animals (e.g., dog, cat), and farm/laboratory animals (e.g., cattle, donkey, goat, horse, pig, sheep; amphibians such as frog, salamander, toad; birds such as chicken, duck, turkey, fishes such as carp, catfish, medaka, salmon, tilapia, tuna, zebrafish; lagomorphs such as hares, rabbits; rodents such as mice, rats).
Stem cells could be maintained and/or maintained in an appropriate niche or in culture, and used as a source of nuclei for cloning progeny organisms via nuclear transfer or a source of cells for propagation of mosaic organisms via embryo aggregation. Thus, Dpp or related BMPs provide a means for growing stem cells in vitro or in vivo for cloning animals.
BMP signaling is unlikely to be confined to one type of BMP and only type of BMP receptor because of the ability of evolutionarily diverged components of the BMP signal transduction pathway or different types of BMPs, BMP receptors, and SMADs to be functional equivalents of each other. For example, there appears to be crosstalk between Dpp/Tkv signaling and Gbb/Sax signaling (Haerry et al., 1998) and one signal transducer acts in different signaling pathways (Lagna et al., 1996). For example, a mixture of BMPs could be added to defined culture medium or be present in conditioned culture medium such that Dpp and Gbb would synergize in initiating BMP signaling through more than one different types of BMP receptor. As another example, one type of signal transducer could stimulate signaling through more than one different types of BMP receptor.
To stimulate BMP signaling, a positive signal transducer could be increased in expression (e.g., more transcripts and/or translated products) or mutated to a gain-of-function phenotype to increase activity of that signal transducer, while a negative signal transducer could be decreased in expression (e.g., fewer transcripts and/or translated products) or mutated to a loss-of-function phenotype to decrease activity of that signal transducer. Alternatively, a downstream target gene of BMP signaling could be directly activated or inhibited without BMP binding to its receptor by genetic engineering using a transactivator like GAL4 binding its UAS or ecdysone receptor binding upstream of the target gene. Similar techniques in mice involve induction with tetracycline or FK506.
Another method would be to increase endogenous BMP activity in the cells or to increase exogenous BMP activity outside the cells, especially if ligand is the limiting component in BMP signaling. For example, BMP expression may be increased in a stem cell and stimulate BMP signaling through an autocrine mechanism. Alternatively, BMP expression may be increased in a non-stem cell or a feeder cell, and then BMP activity could be secreted and taken up by the stem cell or brought into contact with the surface of the stem cell. BMP could also be added to the extracellular space or culture medium. BMP activity may be increased to stimulate BMP signaling by at least about 10%, 50%, 100%, or 200% as compared to the amount normally present in the animal or the culture.
Properties of the stem cell which may be maintained include the following: pluripotency, totipotency, committing to one or more differentiating cell lineages, giving rise to multiple different types of progenitors and/or differentiated cells, contributing to the germline and combinations thereof. Thus, the growth and/or survival of stem cells may be maintained without commitment to a program of differentiation, while retaining the capacity to differentiate normally under appropriate conditions following reduction or elimination of BMP signaling. More simply, stem cells in a population may be expanded in total number or concentration relative to non-stem cells (i.e., an increase in abundance), extended in the time between a stem cell""s birth and its death or apoptosis (i.e., an increase in lifetime), or combinations thereof. Conversely, stem cells or tumor cells in a population may be reduced in total number or concentration, or even eliminated at the limit of detection, by repressing BMP signaling.
Stem cells made according to the present invention may be totipotent or pluripotent, male or female, germline or somatic, dividing or quiescent, vertebrate or invertebrate, present in situ or isolated, partially or substantially purified of differentiate cells, and combinations thereof. Proliferating stem cells are diploid, entering meiosis and the later stages of gametogenesis is part of the program of differentiation for male or female germline stem cells that is prevented by the present invention. Stem cells may be present in or obtained from testis, ovary, especially apical tips of Drosophila testes and/or ovarioles, or other adult or embryonic tissues. By differentiating, stem cells may differentiate into cells of the hematopoietic, immune, or nervous systems or the like. Preferably, stem cells maintained and/or propagated by the present invention retain the potential to later differentiate and thereby contribute to oogenesis or spermatogenesis, all three germ layers (i.e., endoderm, mesoderm, ectoderm), multiple differentiated cell lineages, and combinations thereof.
Somatic cells include terminal filament cells, cap cells, and inner sheath cells from the ovary and hub cells from the testis. Preferably, the present invention reduces the proportion of somatic cells in a population relative to germline cells during maintenance and/or propagation. A niche defined by surrounding somatic cells or a feeder layer comprised of somatic cells may provide cell contact and other extracellular signals to maintain and/or propagate germline cells. A feeder layer may be provided that provides certain essential extracellular signals by, for example, genetically manipulating cultured cells to express and secrete a BMP which then binds to its receptor on the stem cells.
Cell populations may be derived from the germline or somatic (or mixed) male or female, dividing or quiescent,vertebrate or invertebrate, present in situ or isolated, partially or substantially purified, and combinations thereof. Preferably, cell populations include cells expressing one or more BMPs; more preferably, BMP is secreted by non-stem cells and binds to receptors of stem cells to stimulate BMP signaling. Thus, stem cells of the present invention contain receptors for BMP, especially Dpp or a homolog, or are at least responsive to BMP signaling.
Besides mammals, amphibians, birds, and fishes, other organisms may be used in the present invention such as invertebrates like worms (e.g., Helminthes, Nematodes) and insects (e.g., Anopheles, Drosophila). In particular, comparison of components of the BMP signaling pathway, upstream regulators, and downstream targets show them to be highly conserved (Bitgood and McMahon, 1995; Padgett et al., 1998). Thus, the present invention should not be limited in its usefulness to Drosophila melanogaster. Other species which show conservation of dpp (Newfeld et al., 1997) and are likely to be useful are D. simulans, D. pseudoobscura, and D. virilis. For metazoan species in which there has been a diligent search, a dpp-like gene has been identified.
Mammalian homologs of dpp, glass bottom boat (gbb), and screw (scw) have been identified as BMP-2/4, BMP-5/8, and BMP-6, respectively (Hoffmann, 1997; Raftery and Sutherland, 1999; Wharton et al., 1999). A mammalian serine/threonine kinase receptor has been identified that specifically binds BMP-2 and BMP-4 (Yamaji et al., 1994). Other related members of the TGF-xcex2 family, their receptors, or other components of their signaling pathways, might also be used in the present invention. See also U.S. Pat. Nos. 5,011,691, 5,013,649, 5166,058, 5,168,050, 5,216,126, 5,324,819, 5,354,557, 5,635,372, 5,639,638, 5650,276, and 5,854,207.
Furthermore, mutational analysis and determination of structure-function relationships have identified conserved residues and essential residues for Dpp signaling (Wharton et al., 1996). Bacterially expressed Dpp can be refolded, then biochemically and biophysically characterized (Groppe et al., 1998). Homologs of a member of the BMP family, their receptors, and other components of the signaling pathway can be identified by a high level of structural conservation when amino acid sequences are compared, and/or functional conservation when homologs rescue mutant phenotypes or otherwise replace BMP activity.
Cell types have been identified by markers and are well characterized by genetic mutants and developmental studies. Stem cells may be provided in situ as part of an intact organism or they may be cultured in vitro. Germline stem cells and surrounding cells may be from an adult (e.g., ovary, testis) or an embryo. For in vitro culturing, cells may be obtained directly from an organism (i.e., primary culture) but it would be convenient to passage them through several cultures (e.g., at least five, ten, or twenty times) to expand their number (e.g., at least two, ten, or 100 times more than the original number).
Stem cells may be isolated from a donor organism with or without increasing cell number by stimulating BMP signaling; manipulated during transient in vitro culturing under conditions for maintenance and/or propagation by treating with one or more chemicals, introducing genetic material, fusing with another cell, mutating one or more genes, selecting a desired genotype or phenotype, or combinations thereof; and transplanting stem cells back into a host which is identical to the donor (i.e., autologous transplantation), similar to the donor but different (i.e., allogeneic transplantation), or is totally different from the donor (i.e., xenogeneic transplantation). In vitro culture conditions, genetic engineering of Drosophila by transfection and site-specific recombination, and cell or nuclear transplantation are known in the art.
For Drosphila, there are only about 10 germline stem cells per testis and about 32-48 germline stem cells per ovary (i.e., there are about 16 ovarioles per ovary and about two or three germline stem cells per ovary). The present invention provides greatly increased numbers of stem cells to be produced in vivo in an adult or embryo, and then cultured in vitro. In vitro culture of cells may be carried out by initially generating flies with a large number of germline stem cells in each ovariole. Then ovaries may be removed surgically into sterile culture medium and the germ cells released (they do not adhere and, thus, do not need to be disaggregated). Alternatively, disaggregated embryos may also be used as a source of germline stem cells. Although the number of germ cells per embryo is similar to the number per ovary and testis, it is possible to start with 100,000 embryos but only a few hundred gonads can be easily obtained. Schneider (1972) shows derivation of a cell line from Drosophila.
Drosophila cells may be plated into small wells containing feeder layers of cells expressing Dpp (e.g., Panganiban et al., 1990) or Hh (e.g., Lee et al., 1994), or culture media prepared by conditioning the media with cells secreting soluble factors or simply adding a recombinantly produced soluble factor (e.g., Dpp produced according to Groppe et al., 1998). In vitro culture media for growing Drosophila cells are commercially available such as, for example, Schneider""s Drosophila medium. Drosophila cells can also be adapted and grown in mammalian tissue culture media (Spradling et al., 1975; Lengyel et al., 1975). Drosophila cells can be transfected like mammalian cells (Burke et al., 1984). Constructs and strategies for homologous recombination in somatic, embryonic stem (ES), and embryonic (EG) cells could be adapted for use with in vitro cultured Drosophila cells (Capecchi, 1989; Koller and Smithies, 1992). Cultured cells or their nuclei may then be transferred into Drosophila (Okada et al., 1974; Van Deusen, 1977).
Previous attempts at culturing germline stem cells utilized the 40 germline cells present in each embryo at a certain stage of development. But no dpp was provided, and these cells differentiated in culture (Allis et al., 1979). Inducing BMP expression in cells of such cultures or adding exogenous BMP to them would be a simple way of maintaining and/or propagating germline stem cells in vitro.
A BMP may also be used in replacement of, or combination with, known stem growth factors such as, for example, fibroblast growth factor (FGF), leukemia inhibitory factor (LIF), and steel factor (SF). Thus, BMP activity as observed herein might also be demonstrated using the techniques taught in U.S. Pat. Nos. 5,453,357 and 5,690,926.
Ex vivo culturing of stem cells with stimulation of BMP signaling only performed outside the body is preferred to avoid systemic effects of BMP signaling on the organism.
Vascular or organ engineering may be accomplished with stem cells that differentiate into endothelium or parenchyma, respectively, with or without an implantable support (e.g., stent, hollow fiber or particle) on which stem cells have been coated or impregnated. If not autologously transplanted and in an organism with an immune system recognizing histoincompatibility, transplantation of allogeneic or xenogeneic tissue may require immunosuppression of the host (e.g., cyclosporine A or FK506 treatment). Differentiation of stem cells into tissue with the activity and/or structure of adrenal gland, bone marrow, brain, liver, ovary or testis, pancreas, peripheral neurons or glia, red or white blood cells, skeletal or smooth muscle, skin, thyroid gland, or combinations thereof is preferred.
One or more genes of the stem cell may be activated or inhibited by chemical or environmental induction, antisense, ribozyme, chimeric repair vector, RNAi, or random/sequence-specific insertion. Ectopic expression of a gene may be controlled in a particular spatial or temporal manner, mimic pathologic or disease states, or create phenocopies of mutations in the endogenous gene. Homologous recombination is preferred to achieve gene knockout or replacement (see, e.g., U.S. Pat. Nos. 5,569,824, 5,602,307, 5,614,396, 5,683,906, and 5,830,682). For example, stem cells may be transfected with a polynucleotide, the polynucleotide or a portion thereof integrates into the genome of transfected stem cells at a random site or in a sequence-specific manner, homologous recombinants at a genetic loci of interest are selected, and the selected stem cells are transplanted into a host organism. Physical introduction of polynucleotides (e.g., biolistics, electroporation, microinjection) is preferred. Alternatively, insertion of P elements may be genetically engineered in vivo or in vitro in a stem cell maintained and/or propagated according to the present invention to disrupt genes (cf. Zhang and Spradling, 1994; Spradling et al., 1995).
TGF-xcex2 signaling has been shown to limit the growth of germline cysts during Drosophila spermatogenesis (Matunis et al., 1997). When punt or shn function is removed in clones of somatic cells that surround germ cells, cysts continue dividing after four rounds of mitosis (Matunis et al., 1997). However, these investigators did not address whether this pathway functions in male germline stem cells. In the embryo and imaginal discs, punt and shn can function downstream of dpp (Ruberte et al., 1995; Letsou et al., 1995; Arora et al., 1995; Grieder et al., 1995), but it was not known whether dpp or another TGF-xcex2 family member is utilized to send the signal. Clonal analysis of mutants in dpp downstream components in male germline stem cells, like those reported here in the ovary, could show whether Dpp and/or other TGF-xcex2-like molecules are required for their division and maintenance in the testis.
In mouse, the BMP family members BMP-2 and -4 are most closely related to dpp, with greater than 75% identity, and can function to rescue dpp mutants in embryos (Padget et al., 1993). Recently, both genes have been inactivated by homologous recombination, but the homozygous embryos die too early to assess possible functions in the gonads (Winnier et al., 1995; Zhang and Bradley, 1996; reviewed by Hogan, 1996b). Consistent with our findings, Lawson et al. (1999) report that BMP-4 affects the number of primordial germ cells; moreover, BMP4 was needed in somatic tissue, and presumably stimulated BMP signal transduction in germline cells, although this was not shown directly. The roles during spermatogenesis of two other BMP family members, BMP-8A and BMP-8B, have been tested (Zhao et al., 1996; 1998). BMP-8B is required for the resumption of male germline cell proliferation in early puberty, and for germline cell survival in the adult, whereas BMP-8A plays a role in the maintenance of adult spermatogenesis.
The xe2x80x9cnichexe2x80x9d hypothesis postulates that stem cells reside in optimal microenvironments or xe2x80x9cnichesxe2x80x9d (Schofield, 1978). When a stem cell divides, only one daughter can remain in the niche while the other becomes committed to differentiate. A stem cell within the niche would have a high probability of self-renewal, but a low probability of entry into the differentiation pathway. This model is consistent with the observations that stem cells require the addition of growth factors for proliferation and differentiation in many in vitro culture systems (Potter and Loeffler, 1990; Morrison et al., 1997). The molecular nature of the microenvironment within a niche has yet to be defined in any system, although the Drosophila germarium appears to contain such a niche. Anteriorly, the stem cells abut terminal filament and cap cells, which both express hh, while only the latter express armadillo (arm) and wg (Forbes et al., 1996a; 1996b). Stem cell daughters lie more to the posterior, and probably directly contact inner germarial sheath cells, which express patched (ptc) and hh (Forbes et al., 1996b). This asymmetry in structure and signals may allow germline stem cells to receive different levels of signals from their daughters. Consistent with the existence of a niche, two wildtype stem cells in germaria that recently lost a marked mutant stem cell were occasionally observed, suggesting that a vacated niche could be reoccupied.
The existence of the germline stem cell niche is also consistent with stem cell proliferation when local dpp is overexpressed. Under these conditions, the size of the niche may be substantially enlarged. Conversely, reduction of dpp function may weaken the ability of the niche to maintain germline stem cells, leading to accelerated losses. These results suggest that dpp is an essential niche signal. However, dpp likely interacts with other signals from surrounding somatic cells to make a functional niche for germline stem cells. Nonetheless, the identification of dpp as a key niche signal should greatly facilitate efforts to culture Drosophila germline stem cells in vitro.
Technical limitations have previously prevented identification of the source of the dpp signal that is received by germline stem cells. Ideally, analysis of clones of a null dpp allele would reveal which cells produce the signal. However, the somatic cells adjacent to the stem cells cease division early in ovary development and make induction of specific small clones difficult. The pattern of dpp expression in the germarium should also provide some insight into the origins of the signal. However, the only available dpp-lacZ fusion line and whole mount in situ experiments failed to detect-expression in the germarium, although follicle cell expression in late stage egg chambers was observed. We now show that somatic cells in the niche express dpp. In many systems, low levels of dpp expression are known to be sufficient for biological effects so it may be sufficient to provide only low levels of BMP in the present invention.
In the Drosophila leg, antenna and genital discs, dpp and wg are induced in the anterior compartment by hh, and the mutual repression of dpp and wg restricts them to their appropriate domains (Brook and Cohen, 1996; Jiang and Struhl, 1996; Chen and Baker, 1996). In vertebrate limb development, sonic hedgehog (shh) can induce the expression of BMP-2 (Johnson and Tabin, 1995). The somatic terminal filament, cap, and inner sheath cells express hh and lie adjacent to the germline stem cells (Forbes et al., 1996a, 1996b). wg and dpp expression may be induced by hh, and signal to germline stem cells for their proliferation and maintenance. The data indicate that these and possibly additional signals from the anterior somatic cells define a niche for germline stem cells at the tip of germarium. Thus, agents which modify hedgehog signaling may be used to alter local BMP signaling, thereby regulating stem cell maintenance and/or propagation.